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Background  Diphtheria toxin (DT) is produced by toxigenic strains of the human pathogen  Corynebacterium diphtheriae  as well as zoonotic  C. ulcerans  and  C. pseudotuberculosis . Toxigenic strains may cause severe respiratory diphtheria, myocarditis, neurological damage or cutaneous diphtheria. The DT encoding  tox  gene is located in a mobile genomic region and  tox  variability between  C. diphtheriae  and  C. ulcerans  has been postulated based on sequences of a few isolates. In contrast, species-specific sequence analysis of the diphtheria toxin repressor gene ( dtxR ), occurring both in toxigenic and non-toxigenic  Corynebacterium  species, has not been done yet. We used whole genome sequencing data from 91 toxigenic and 46 non-toxigenic isolates of different pathogenic  Corynebacterium  species of animal or human origin to elucidate differences in extracted DT, DtxR and  tox -surrounding genetic elements by a phylogenetic analysis in a large sample set.    Results  Sequences of both DT and DtxR, extracted from whole genome sequencing data, could be classified in four distinct, nearly species-specific clades, corresponding to  C. diphtheriae ,  C. pseudotuberculosis ,  C. ulcerans  and atypical  C. ulcerans  from a non-toxigenic toxin gene-bearing wildlife cluster. Average amino acid similarities were above 99% for DT and DtxR within the four groups, but lower between them. For DT, subgroups below species level could be identified, correlating with different  tox -comprising mobile genetic elements. In most  C. diphtheriae ,  tox  genes were located within known prophages. In contrast, in  C. ulcerans  diverse  tox -including mobile elements could be identified: either prophages differing from  C. diphtheriae  prophages or an alternative pathogenicity island (PAI) described previously. One isolate showed a different, shorter  tox -comprising putative PAI. Beyond the  tox -overlapping elements, most isolates harbored a variety of additional prophages.    Conclusion  Our NGS data from 137 isolates indicate the existence of different genetic backgrounds of DT-mediated pathogenicity in different  Corynebacterium  species and evolution of once acquired pathogenicity features with the strains. Different groups of pathogenicity-related elements within  C. ulcerans  imply that  tox  transmission pathways between isolates may differ in the zoonotic species and contribute to their emerging pathogenic potential.    Electronic supplementary material  The online version of this article (10.1186/s12866-019-1402-1) contains supplementary material, which is available to authorized users.

NGS-based phylogeny of diphtheria-related pathogenicity factors in different Corynebacterium spp. implies species-specific virulence transmission